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Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

Objectives: Muscle release of the amino acid 3-methyl-histidine (3MH) is a sensitive index of myofibrillar protein overdegradation (MPO). We hypothesized that patients with chronic heart failure (CHF) could have increased muscle release of 3MH, which in turn reflects MPO, and that serum electrolyte sodium (Na(+)) and potassium (K(+)) levels may be associated with this 3MH muscle release., Methods: Thirty-one overweight outpatients (body mass index, 27 ± 4.4 kg/m(2); 22 men and 9 women; age, 56 ± 8.7 y) with clinically stable CHF were studied. After a 24-hour meat-free diet and overnight fasting, patients underwent blood sampling from a cannulated arm vein (V) and concomitantly from the arterial artery (A) to determine plasma 3MH levels and to calculate the A-V difference. Serum levels of Na(+) and K(+) in the venous blood were determined, and the Na(+)/K(+) ratio was calculated. Ten healthy subjects who were matched for gender, age, and body mass index served as controls and underwent the same protocol as the patients with CHF., Results: The patient group had higher arterial (P = 0.02) and venous (P = 0.005) 3MH levels but a similar A-V 3MH difference (P = 0.28) as compared with the controls. Within the CHF group, 67.7% of patients released 3MH, which resulted in a negative A-V value (P < 0.02 as compared with controls). In patients with CHF, the A-V 3MH difference correlated positively with the serum K(+) level (r = 0.62; P = 0.0002) and negatively with Na(+)/K(+) ratio (r = -0.55; P = 0.002). No association was found between the A-V 3MH difference and the Na(+) level., Conclusions: The study demonstrated the existence of MPO in resting overweight patients with CHF, thereby suggesting that low serum levels of K(+) may contribute to MPO., (Copyright © 2014 Elsevier Inc. All rights reserved.)

It is unclear whether the muscle hypertrophy induced by loss of myostatin signaling in mature muscles is maintained only by increased protein synthesis or whether reduced proteolysis contributes. To address this issue, we depleted myostatin by activating Cre recombinase for 2 wk in mature mice in which Mstn exon 3 was flanked by loxP sequences. The rate of phenylalanine tracer incorporation into myofibrillar proteins was determined 2, 5, and 24 wk after Cre activation ended. At all of these time points, myostatin-deficient mice had increased gastrocnemius and quadriceps muscle mass (≥27%) and increased myofibrillar synthesis rate per gastrocnemius muscle (≥19%) but normal myofibrillar synthesis rates per myofibrillar mass or RNA mass. Mean fractional myofibrillar degradation rates (estimated from the difference between rate of synthesis and rate of change in myofibrillar mass) and muscle concentrations of free 3-methylhistidine (from actin and myosin degradation) were unaffected by myostatin knockout. Overnight food deprivation reduced myofibrillar synthesis and ribosomal protein S6 phosphorylation and increased concentrations of 3-methylhistidine, muscle RING finger-1 mRNA, and atrogin-1 mRNA. Myostatin depletion did not affect these responses to food deprivation. These data indicate that maintenance of the muscle hypertrophy caused by loss of myostatin is mediated by increased protein synthesis per muscle fiber rather than suppression of proteolysis.

Oral contraceptive (OC) treatment has an inhibiting effect on protein synthesis in tendon and muscle connective tissue. We aimed to investigate whether OC influence myofibrillar protein turnover in young women. OC-users (24±2 years; Lindynette® n=7, Cilest® n=4) and non-OC-users (controls, 24±4 years n=12) performed one-legged kicking exercise. The next day, the myofibrillar protein fractional synthesis rate (FSR) was measured using stable isotopic tracers ((13)C-proline) while the subjects were fed standardized nutrient drinks. Simultaneously, a marker for myofibrillar protein breakdown, 3-methyl-histidine (3-MH), was measured in the interstitial fluid of the vastus lateralis. Measurements were performed in both legs. In general, myofibrillar protein FSR was lower in OC-users (two-way analysis of variance, P<0.05), although the difference seemed to depend on the OC type. Interstitial 3-MH in the skeletal muscle was not different between groups and did not vary by OC type. Exercise did not change myofibrillar protein FSR or 3-MH concentrations. Serum androstenedione and bioavailability of testosterone were lower in OC-users. In conclusion, the results indicate that the use of OC has an inhibiting effect on myofibrillar protein synthesis and the magnitude of the effect may depend on the type of OC. In contrast, there was no effect of OC on myofibrillar protein breakdown in the fed state., (© 2009 John Wiley & Sons A/S.)

The physiologic response to trauma results in the efflux of large amounts of amino acids from skeletal muscle. This is extreme in large burn injuries. Protein kinetic studies, although useful in determining the rates of protein synthesis and breakdown, do not provide information about muscle loss. This study determined the contribution of muscle protein to whole-body protein breakdown in children throughout their course of burn injury. Children aged 0 to 18 years with initial burn size ≥30% TBSA underwent ¹⁵N glycine and 3 methylhistidine (3MH) analysis during three phases of care: A, early acute; B, wound closure; and C, convalescence. Muscle protein breakdown was estimated using a factor of 4.2 μmol 3MH per 1 g of mixed protein. Twenty-two patients with a mean of 54.5 ± 20.1% TBSA burn were studied. Protein balance did not change remarkably and remained positive by 2 g/kg during hospitalization. However, muscle protein breakdown dropped from 1.1 to 0.6 g/kg with wound closure (P < .0001), representing a decrease in the contribution of muscle protein to whole-body protein breakdown from 20 to 7%. Ten patients returned for a third measurement after discharge. Although protein turnover was high, muscle breakdown was consistent with 3MH values reported in healthy children. Serial determination of 3MH excretion is a simple way to track muscle catabolism throughout burn injury. Our data suggest that despite accelerated protein turnover, muscle catabolism significantly decreases with wound closure and begins to normalize around discharge. In convalescence, 3MH excretion is comparable with normal children.

Skeletal muscle atrophy is evident after muscle disuse, unloading, or spaceflight and results from decreased protein content as a consequence of decreased protein synthesis, increased protein breakdown or both. At this time, there are essentially no human data describing proteolysis in skeletal muscle undergoing atrophy on Earth or in space, primarily due to lack of valid and accurate methodology. This particular study aimed at assessing the effects of short-term unloading on the muscle contractile proteolysis rate. Eight men were subjected to 72-h unilateral lower limb suspension (ULLS) and intramuscular interstitial levels of the naturally occurring proteolytic tracer 3-methylhistidine (3MH) were measured by means of microdialysis before and on completion of this intervention. The 3MH concentration following 72-h ULLS (2.01 +/- 0.22 nmol/ml) was 44% higher (P < 0.05) than before ULLS (1.56 +/- 0.20 nmol/ml). The present experimental model and the employed method determining 3MH in microdialysates present a promising tool for monitoring skeletal muscle proteolysis or metabolism of specific muscles during conditions resulting in atrophy caused by, e.g., disuse and real or simulated microgravity. This study provides evidence that the atrophic processes are evoked rapidly and within 72 h of unloading and suggests that countermeasures should be employed in the early stages of space missions to offset or prevent muscle loss during the period when the rate of muscle atrophy is the highest.

Objective: To study the changes of skeletal muscle proteolysis in severely burned patients with sepsis and analyze its possible mechanism., Methods: Blood samples were collected from 20 burned patients with sepsis, aged 42 +/- 10 (burn sepsis group), and 12 patients undergoing plastic operation after burn, aged 43 +/- 8 (control group) to determine the plasma concentrations of cortisol and tumor necrosis factor (TNF-alpha) by radioimmunoassay. 24 h urine was retained to determine the level of 3-methylhistidine (3-MH) by high performance liquid chromatography. An appropriate amount of quadriceps femoris muscle sample was collected during operation to determine the concentration of 3-methylhistidine with high performance liquid chromatography, the expression of ubiquitin mRNA, E(2)-14k mRNA and C(2) subunit mRNA by Northern blot analysis, and the protein expression of C(2) subunit by Western blot analysis., Results: The plasma concentrations of cortisol and TNF-alpha in the burn sepsis group were 478 +/- 56 microg/L and 22.4 +/- 3.8 microg/L respectively, both significantly higher than the those in the normal control group, 72 +/- 12 microg/L and 0.5 +/- 0.1 microg/L respectively (both P < 0.01). The amount of 3-MH in the quadriceps femoris muscle of the burn sepsis group was 4.3 +/- 0.6 micromol/g, significantly higher than that of the control group (2.5 +/- 0.4 micromol/g, P < 0.01). The 24 h urinary output of 3-MH of the burn sepsis group was 456 +/- 25 micromol, significantly higher than that of the control group (202 +/- 29 micromol, P < 0.091). The expressions of ubiquitin mRNA 2.4 Kilobase pair (kb) and 1.2 kb, E(2)-14k mRNA 1.2 kb and C(2) subunit mRNA in quadriceps femoris muscles of the burn sepsis group were increased with 51%, 32%, 75%, and 39% respectively, as compared with those of the control group (all P < 0.01). The protein expression of C(2) subunit in quadriceps femoris muscles of the burn sepsis group was increased with 130% than that of the control group (P < 0.01)., Conclusion: The proteolytic rate in skeletal muscle is increased during sepsis after severe burn and the mechanism is related to the enhanced function of the ubiquitin system at both gene and protein levels with the hypersecretion of glucocorticoid and TNF-alpha.

Purpose of Review: To describe the latest innovations in measuring protein breakdown in vivo, particularly in muscle., Recent Findings: The traditional method of using 3-methylhistidine excretion to measure muscle protein breakdown has been updated to include arteriovenous or microdialysis measurements, which address the concern that there are alternative sources of 3-methylhistidine in the body other than muscle. Several variations of a precursor-product method to measure fractional breakdown rate of tissues have been developed that are analogous to fractional synthesis rate of tissues. These methods are more generally applicable than the 3-methylhistidine methods and are less invasive than arteriovenous methods. The various precursor-product methods are distinguished by whether they require an isotopic steady state or multiple tracers and by how many biopsies are required., Summary: The new precursor-product methods have enabled assessment in clinical trials of protein breakdown for proteins other than myofibrillar proteins and in circumstances in which arteriovenous sampling is not feasible.

Muscle atrophy results primarily from accelerated protein degradation and is associated with increased expression of two muscle-specific ubiquitin ligases (E3s): atrogin-1 and muscle ring finger 1 (MuRF1). Glucocorticoids are essential for many types of muscle atrophy, and their effects are opposite to those of insulin-like growth factor I (IGF-I) and insulin, which promote growth. In myotubes, dexamethasone (Dex) inhibited growth and enhanced breakdown of long-lived cell proteins, especially myofibrillar proteins (as measured by 3-methylhistidine release), while also increasing atrogin-1 and MuRF1 mRNA. Conversely, IGF-I suppressed protein degradation and prevented the Dex-induced increase in proteolysis. IGF-I rapidly reduced atrogin-1 expression within 1 h by blocking mRNA synthesis without affecting mRNA degradation, whereas IGF-I decreased MuRF1 mRNA slowly. IGF-I and insulin also blocked Dex induction of these E3s and several other atrophy-related genes ("atrogenes"). Changes in overall proteolysis with Dex and IGF-I correlated tightly with changes in atrogin-1 mRNA content, but not with changes in MuRF1 mRNA. IGF-I activates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, and inhibition of this pathway [but not the calcineurin-nuclear factor of activated T cell (NFAT) or the MEK-ERK pathway] increased proteolysis and atrogin-1 mRNA expression. Thus an important component of growth stimulation by IGF-I, through the PI3K-Akt pathway, is its ability to rapidly suppress transcription of the atrophy-related E3 atrogin-1 and other atrogenes and degradation of myofibrillar proteins.

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation after refeeding in young rats and mice. However, it is unclear whether this suppression is seen in adult animals. This study was undertaken to compare dietary protein-induced suppression of myofibrillar protein degradation in young and adult mice. Reductions in rates of myofibrillar protein degradation measured by N-methylhistidine (MeHis) released from the isolated extensor digitorum longus muscle were found at 4 to 7 h after refeeding in both young (7-wk-old) and adult (8-mo-old) mice, indicating that the response time of feeding-induced suppression of myofibrillar protein degradation was the same. When young (8-wk-old) mice were fed a 20% casein diet (20C) for 1 h after 18 h starvation, the rate of myofibrillar protein degradation was significantly decreased at 4 h after refeeding; however, mice fed a 10% casein diet (10C), 5% casein diet (5C), or protein-free diet (0C) did not show suppression of myofibrillar protein degradation. Adult (8-mo-old) mice fed 20C or 10C showed a reduction in the rate of MeHis release. The plasma concentration of leucine in young mice was only higher when they were fed 20C. Adult mice fed 20C or 10C showed higher plasma concentrations of leucine. These results suggest that postprandial suppression of myofibrillar protein degradation occurs in adult mice as in young mice, but the adult mice respond to a lower amount of dietary casein compared to the young mice.

The actions of the thyroid hormone (T3) are modified by other hormones. Therefore, in the normal cell culture system, with serum as a medium ingredient, it is difficult to eliminate the influences of other hormones derived from the serum. In the present study, two experiments were conducted to clarify the effects of T3 on differentiation, growth and proteolysis (experiment 1), and concerted effects of T3 and insulin (experiment 2) in a serum-free culture with the use of muscle cells originated from chick embryo. Protein content and creatine kinase (CK) activity were examined as indices of growth and differentiation of the muscle cells, respectively, and N(tau)-methylhistidine (MeHis) release was examined as an index of myofibrillar proteolysis. It was observed that T3 suppressed both the muscle differentiation and proteolysis in the serum-free medium (experiment 1), though in our previous experiment they were enhanced by T3 in the serum-supplemented normal medium. On the other hand, T3 increased myofibrillar proteolysis and had no effect on muscle differentiation when insulin was included in the serum-free medium (experiment 2). These results clearly show that the effects of thyroid hormones on muscle differentiation and proteolysis are apparently different when serum is deprived from the medium, and these differential effects of thyroid hormone could be partially explained by an interaction with insulin, one of the growth factors in the serum.

Objectives: To study the effect of a parenteral nutrition solution enriched with potential precursors of glutamine, i.e., arginine and glutamate, on plasma glutamine concentrations and protein metabolism., Design: Prospective, randomized, single-blind, comparative study., Setting: Two intensive care units in two different hospitals., Patients: Fifteen surgical patients., Interventions: Patients were randomized to receive total parenteral nutrition for 5 days with the enriched glutamine precursor solution (GlnP+ group) or a conventional solution (control group), both total parenteral nutrition providing 0.25 gN/kg per day and 35 kcal/kg per day (glucose/lipids, 70%:30%)., Measurements and Main Results: Plasma amino acid concentrations before (T0) and after 3 hrs (T3) of perfusion, nitrogen balance (daily and cumulated), and urinary excretion of 3-methylhistidine were measured daily from day 1 to day 5. The two groups were identical for age, weight, severity score, and nitrogen and energy intakes. After a 3-hr perfusion, plasma concentrations of arginine, ornithine, and glutamine increased, and the differences (T3 - T0) were significantly higher in the GlnP+ group: arginine, 107.6+/-7.0 vs. 51.9+/-3.3 (mean over 5 days; p < .001); ornithine, 78.9+/-7.1 vs. 43.6+/-3.1 (p < .001); and glutamine, 32.4+/-8.6 vs. 6.7+/-5.0 micromol/L (p < .05), respectively. A positive correlation was found between arginine and glutamine plasma increases only in the GlnP+ group: r = .45; p < .01 (Spearman's rank-correlation test). Daily and cumulated nitrogen balances were not significantly different between the two groups but were positive (difference from 0) only in the GlnP+ group. The urinary 3-methylhistidine/creatinine ratio decreased significantly from day 1 to day 5 only in the GlnP+ group: 24.5+/-2.7 vs. 18.8+/-2.7 micromol/mmol (p < .05)., Conclusions: Total parenteral nutrition enriched with arginine and glutamate promotes a better nitrogen balance, limits protein myofibrillar catabolism, and generates glutamine, with arginine (not glutamate) probably being the main contributor to the glutamine-generating effect of the solution through the formation of ornithine.

Eighteen pure-bred steers (live weight 350 kg) from each of two breeds, Aberdeen Angus (AA) and Charolais (CH), were split into three equal groups (six animals each) and offered three planes of nutrition during a 20-week period. The same ration formulation was offered to all animals with amounts adjusted at 3-week intervals to give predicted average weight gains of either 1.0 kg/d (M/M group) or 1.4 kg/d (H/H group). The remaining group (M/H) were offered the same amount of ration as the M/M group until 10 weeks before slaughter when the ration was increased to H. Data on animal performance, carcass characteristics and fibre-type composition in skeletal muscle are presented elsewhere (Maltin et al. 2000; Sinclair et al. 2000). On three occasions (17, 10 and 2 weeks before slaughter) the animals were transferred to metabolism stalls for 1 week, during which total urine collection for quantification of Ntau-methylhistidine (Ntau-MeH) elimination was performed for 4 d. On the last day, animals were infused for 11 h with [2H5]phenylalanine with frequent blood sampling (to allow determination of whole-body phenylalanine flux) followed by biopsies from m. longissimus lumborum and m. vastus lateralis to determine the fractional synthesis rate of mixed muscle protein. For both breeds, the absolute amount of Ntau-MeH eliminated increased with animal age or weight (P < 0.001) and was significantly greater for CH steers, at all intake comparisons, than for AA (P < 0.001). Estimates of fractional muscle breakdown rate (FBR; calculated from Ntau-MeH elimination and based on skeletal muscle as a fixed fraction of live weight) showed an age (or weight) decline for M/M and H/H groups of both breeds (P < 0.001). FBR was greater for the H/H group (P = 0.044). The M/H group also showed a lower FBR for the first two measurement periods (both at M intake) but increased when intake was raised to H. When allowance was made for differences in lean content (calculated from fat scores and eye muscle area in carcasses at the end of period 3), there were significant differences in muscle FBR with intake (P = 0.012) but not between breed. Whole-body protein flux (WBPF; g/d) based on plasma phenylalanine kinetics increased with age or weight (P < 0.001) and was similar between breeds. The WBPF was lower for M/M compared with H/H (P < 0.001) based on either total or per kg live weight0.75. Muscle protein fractional synthesis rate (FSR) declined with age for both breeds and tended to be higher at H/H compared with M intakes (intake x period effects, P < 0.05). Changing intake from M to H caused a significant increase (P < 0.001) in FSR. The FSR values for AA were significantly greater than for CH at comparable ages (P = 0. 044). Although FSR and FBR responded to nutrition, these changes in protein metabolism were not reflected in differences in meat eating quality (Sinclair et al. 2000).

Induction of experimental insulin-deficiency by a single administration of streptozotocin to rats resulted in substantial changes in heart and skeletal muscle size and protein content. This was accompanied by a marked loss of total body (carcass) nitrogen and raised concentrations of circulating branched-chain amino acids. These changes were related to alterations in protein turnover in skeletal muscle. Thus, the diabetic animals showed changes in both the fractional protein rates of synthesis (decreased by 37%) and degradation (increased by 141%). The increased protein degradation observed in the muscle of the diabetic animals was associated only with an increase in the expression of the genes controlling ubiquitin-dependent proteolysis. It may be suggested that the hormonal changes associated with the diabetic state play an important role in the regulation of the activity of the ubiquitin-dependent proteolytic system in skeletal muscle, highlighting the major role of this system in the diabetes-related cachexia.

Sepsis is associated with reduced protein synthesis and increased protein degradation in skeletal muscle. We examined the effects of insulin-like growth factor I (IGF-I) on protein synthesis and breakdown in muscles from nonseptic and septic rats. Sepsis was induced by cecal ligation and puncture; control rats were sham operated. Extensor digitorum longus muscles were incubated in the absence or presence of IGF-I at concentrations ranging from 100 ng/ml to 10 micrograms/ml. Total and myofibrillar protein breakdown rates were measured as net release of tyrosine and 3-methylhistidine, respectively. Protein synthesis was determined by measuring incorporation of [U-14C]phenylalanine into protein. IGF-I stimulated protein synthesis in a dose-dependent fashion in muscles from both sham-operated and septic rats, with a maximal effect seen at a hormone concentration between 500 and 1,000 ng/ml. IGF-I inhibited total and myofibrillar protein breakdown in muscles from sham-operated rats, whereas in muscles from septic rats, IGF-I had no effect on protein breakdown, even at high concentrations. The results suggest that protein breakdown in skeletal muscle becomes resistant to IGF-I during sepsis and that this resistance reflects a postreceptor defect.

Measurement of urinary 3-methylhistidine (3MH) excretion is the primary in vivo method to measure skeletal muscle (myofibrillar) protein breakdown. This method requires quantitative collection of urine and is based on the assumption that no metabolism of 3MH occurs once it is released from actin and myosin. This is true in most species, but in sheep and swine a proportion is retained in muscle as a dipeptide, balenine. In neither of these species does urine 3MH yield any data on the metabolism of 3MH. We have conducted studies that propose that 3MH metabolism in humans, cattle, dogs, swine, and sheep can be defined from a single bolus infusion of a stable isotope 3-[methyl-2H3]-methylhistidine. Following the bolus dose of the stable isotope tracer, serial blood samples and/or urine was collected over three to five days. A minimum of three exponentials were required to describe the plasma decay curve adequately. The kinetic linear-time-invariant models of 3MH metabolism in the whole animal were constructed by using the SAAM/CONSAM modeling program. Three different configurations of a three-compartment model are described: (A) A simple three-compartment model for humans, cattle, and dogs, in which plasma kinetics (3-[methyl-2H3]-MH/3MH) are described by compartment 1 and with one urinary exit from compartment 1. (B) A plasma-urinary kinetic three-compartment model with two exits was used for sheep with a urinary exit out of compartment 1 and a balenine exit out of a tissue compartment 3. (C) A plasma three-compartment model was used in swine with an exit out of a tissue compartment 3. The kinetic parameters reflect the differences in known physiology of humans, cattle, and dogs as compared to sheep and swine that do not quantitatively excrete 3MH into the urine. Steady-state model calculations define masses and fluxes of 3MH between three compartments and, importantly, the de novo production of 3MH. The de novo production of 3MH for humans, cattle, dogs, sheep, and swine are 3.1, 6.0, 12.1, 10.3, and 7.2 mumol x kg-1 x d-1, respectively. The de novo production of 3MH as calculated by the compartmental model was not different when compared to 3MH production as calculated via traditional urinary collection. Additionally, data suggest that steady-state compartment masses and mass transfer rates may be related to fat free mass and muscle mass in humans and swine, respectively. In conclusion, models of 3MH metabolism have been developed in numerous species, and these models can be used for the assessment of muscle proteolysis and 3MH kinetics without the collection of urine. This methodology is less evasive and will be useful in testing further experimental designs that alter myofibrillar protein breakdown.

A study was conducted to clarify the contribution by myofibrillar protein to whole-body protein breakdown in surgically stressed patients. Thirteen patients who underwent esophagectomy (group E) and 22 who underwent gastric or colorectal operation (group GC) were studied. Patients were all male and younger than 65 y old. Whole-body protein breakdown was determined using constant infusion of 15N-glycine. Urinary excretion of total catecholamines and 3-methylhistidine (3-MH) were measured. Amino acid composition of femoral arterial and venous blood was also analyzed. All the patients were fed exclusively by total parenteral nutrition providing 1.5 g protein and 40 kcal.kg-1.d-1 throughout the study. Whole-body protein breakdown increased significantly in group E (P < 0.01) and group GC (P < 0.05) on the 3rd postoperative day. The increase was significantly greater in group E than group GC (P < 0.01). Urinary excretion of 3-MH also increased significantly in group E (P < 0.01) and in group GC (P < 0.01) on the 3rd postoperative day. The increase was also greater in group E than group GC (P < 0.01). The ratio of urinary 3-MH excretion to whole-body breakdown protein (mumol/g), which is a indicator for the contribution of myofibrillar protein to the whole-body protein breakdown, increased significantly from 0.84 +/- 0.30 of preoperative value to 1.79 +/- 0.38 in group E (mean +/- SD; P < 0.01) and 1.42 +/- 0.18 in group GC (P < 0.05) on the 3rd postoperative day. This ratio was significantly higher in group E (P < 0.05). Furthermore, the ratio of myofibrillar to whole-body protein breakdown correlated significantly with urinary excretion of total catecholamines (r = 0.546; P < 0.01). Therefore, the contribution of myofibrillar protein to whole-body protein breakdown increased proportionately with the severity of surgical stress. On the other hand, femoral-arteriovenous differences of BCAA, Ala, Gln, Tyr, and Phe correlated significantly with the urinary excretion of 3-MH. These data suggest that skeletal muscle protein degradation is proportional to the breakdown of total myofibrillar proteins and both correlate with the severity of stress. From these data, it may be suggested that the contribution of skeletal muscle to whole-body protein catabolism is increased postoperatively, and that the increase is correlated with the severity of surgical stress.

The rates of [3H]N(tau)-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [3H]methionine, were used to measure myofibrillar protein degradation. In fused C2C12 myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions +/- leucine, but in the absence of [3H]-methionine, rates of protein accretion and synthesis over 24-48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types.

In normal muscle there is a delicate balance between muscle protein synthesis and protein degradation. It is believed that this balance is disturbed in muscular dystrophy (MD) by decreased muscle protein synthesis and/or increased muscle protein degradation, resulting in net catabolism. In an attempt to reduce or reverse this catabolism, a high protein diet (HPD, 50% protein) was fed to dystrophic mice (129/ReJ dy) for 4 wk. The effects on muscle biochemistry, muscle function and muscle morphology were compared with those in dystrophic mice fed a normal diet (NPD, 20% protein) and in nondystrophic mice (NORM) also fed the 20% protein diet. Compared with NORM mice, NPD mice demonstrated greater rates of muscle protein synthesis (P < 0.05) as measured by the incorporation of labeled phenylalanine into muscle, greater protein degradation (P < 0.01) as measured by urinary 3-methylhistidine excretion, and lower muscle protein concentration (P < 0.01). When dystrophic mice were fed HPD for 4 wk, protein degradation was lower (P < 0.01) and muscle protein concentration greater (P < 0.01) than in NPD mice. These biochemical improvements were accompanied by greater morphological uniformity of muscle fibers, higher volume density of muscle fibers per unit area of muscle (P < 0.01), and lower shape factor (P < 0.01). Functionally, HPD led to improved muscle endurance (P < 0.01) and increased hind-limb utilization (P 0.01). We conclude that in murine dystrophy, HPD decreases net muscle catabolism, principally by decreasing muscle protein degradation, resulting in improvement in muscle morphology, strength and function.

1. The role of interleukin-1 (IL-1) in sepsis-induced muscle proteolysis was assessed by treating septic rats with recombinant IL-1 receptor antagonist (rIL-1ra). 2. In initial experiments, we tested the effectiveness of IL-1ra in preventing muscle proteolysis induced by administration of IL-1. 3. When normal rats were treated with rIL-1 alpha (three intraperitoneal doses of 100 micrograms/kg body weight each over 16 hr), total and myofibrillar muscle protein breakdown rates, measured as release of tyrosine and 3-methylhistidine, respectively, by incubated extensor digitorum longus muscles, were significantly increased. 4. This metabolic response to IL-1 alpha was completely abolished by rIL-1ra, administered as three intraperitoneal doses of 3 mg/kg body weight each over 16 hr. 5. In subsequent experiments, sepsis was induced in rats by cecal ligation and puncture (CLP); non-septic rats were sham-operated. 6. Treatment of septic rats over 16 hr with a total dose of 25 mg/kg body weight of rIL-1ra reduced, but did not normalize, the increased muscle protein breakdown rates seen during sepsis. 7. When the dose of rIL-1ra was more than doubled and given as a constant infusion at a rate of 4.2 mg/kg body weight/hr for 16 hr, the increased rate of muscle proteolysis in septic rats was normalized. 8. The present study offers the first direct evidence that IL-1 is involved in the regulation of muscle proteolysis during sepsis.

In previous studies, interleukin-1 (IL-1) or tumor necrosis factor (TNF) have been demonstrated to augment skeletal muscle protein breakdown in a manner similar to that induced by bacterial endotoxin. This response to IL-1 or TNF was elicited only after they were administered to animals for various periods, as their addition in vitro to incubated muscles from normal untreated rats was without effect. This suggested that IL-1 and TNF may augment muscle proteolysis in an indirect fashion. Serum levels of IL-1, TNF as well as interleukin-6 (IL-6) are all elevated during infection induced by bacterial endotoxin. Both IL-1 and TNF can induce the synthesis of IL-6 by a variety of cells. Because of this, in the present report, the ability of IL-6 to stimulate skeletal muscle protein breakdown was examined. Muscle protein breakdown was evaluated by measuring the release of both tyrosine and 3-methylhistidine by incubated muscles. Pretreatment of rats with IL-6 for 6 hr induced fever and increased the release of both tyrosine and 3-methylhistidine by the extensor digitorum longus muscle. However, IL-6 did not augment muscle proteolysis when muscles from normal untreated rats were incubated in the presence of the cytokine. The data suggest that the acute treatment of animals with IL-6 can augment muscle proteolysis. Whether this response is due to a direct effect of IL-6 on the myocyte or whether it is due to the production of other mediators remains unclear.

The purpose of this study was to examine the effect of exercise on the rate of protein degradation in rat skeletal muscle. The rates of total and myofibrillar protein degradation were determined by the measurement of the rates of release of tyrosine and 3-methylhistidine, respectively, from the perfused single rat leg. This method measures the rate of protein degradation in the entire lower leg and does not suffer from the limitations inherent in methods that rely on urinary excretion. The rate of total protein degradation was increased by exercise and involved increased flux through the lysosomal pathway, while the breakdown of myofibrillar protein was unchanged. The changes in the rates of protein degradation during the recovery period were greatly influenced by energy intake. Again the rate of myofibrillar protein degradation was unchanged or slightly increased during the recovery period, after either level or downgrade running. Exercise did prevent the increase in the rate of total protein degradation caused by food restriction, which may have important implications in weight reduction diets.

In this study we compared the effect of sepsis on muscle protein metabolism in infant (3 to 4 weeks) and adult (3 to 4 months) rats. Sepsis was induced by cecal ligation and puncture (CLP). Control animals underwent sham operation. Sixteen hours after CLP or sham operation, metabolic studies were performed in incubated intact extensor digitorum longus muscles from infant rats or in strips of the same muscle from adult rats. Protein synthesis rate was determined as incorporation of 3H-phenylalanine into protein; total and myofibrillar protein breakdown rates were determined as release of tyrosine and 3-methylhistidine, respectively. Mortality rate following CLP was similar in both age groups. Basal protein synthesis rate was 3 times higher, total protein breakdown rate was 50% higher, and myofibrillar protein breakdown rate was 3 times higher in infant than in adult animals. However, the relative changes in protein turnover rates induced by sepsis were similar in infant and adult rats: protein synthesis rate decreased by approximately 30%, total protein breakdown increased by 40% to 50%, and myofibrillar protein breakdown increased severalfold. The data suggest that despite prominent differences in basal protein turnover rates between infant and adult rats, the effect of sepsis on muscle protein metabolism is not age dependent.

Urinary N tau-methylhistidine (NMH) excretion has been used as an index of muscle protein breakdown in cattle. An alternative means to estimate muscle proteolysis in cattle is to estimate the de novo production of NMH from plasma kinetics isotopically. Three crossbred steers (average 229 kg) were given a 5.0-mg bolus intravenous injection of [methyl-2H3-N tau-methylhistidine (d3-NMH), after which 16 serial blood samples and three consecutive 24-h urine samples were taken. The enrichment of NMH in plasma was determined by gas chromatography-mass spectrometry, and compartmental analysis of the kinetic data was performed using the SAAM modeling program. The NMH production rates per day (NMHPR, micromoles per day) were 732, 782, and 725, and the fractional breakdown rates (FBR, percentage per day) were 1.61, 1.72, and 1.58 as determined by urinary excretion of NMH, by a three-pool catenary model (plasma kinetics, Model A), and by a more descriptive, three-pool model with two response curves (both plasma and urine kinetics, Model B), respectively. Model A and B estimates of NMHPR and FBR were similar (P greater than .25) to those of estimates obtained from urinary NMH excretion. Kinetic modeling also allows calculation of compartment mass and flux of NMH between compartments and indicates that when NMH exists the muscle pool it is rapidly excreted via the urine. In conclusion, kinetic modeling offers an alternative approach to estimating the NMH production rate.

Daily treatment of Fischer-344 rats for 14 days with the beta 2-adrenergic agonist, clenbuterol, increased gastrocnemius muscle mass and protein content. Coadministration with the beta-adrenergic antagonist, nadolol, significantly reduced these anabolic effects of clenbuterol. Although clenbuterol treatment reduced food intake during the first 4 days, clenbuterol-treated rats were hyperphagic during the second week of drug administration. Nadolol treatment also blocked these effects of clenbuterol on feeding. In a second experiment, in vitro incubation of extensor digitorum longus muscles taken from post weaning food-deprived rats demonstrated decreased release of 3-methylhistidine by clenbuterol-treated rats, suggesting decreased breakdown of myofibrillar protein. Protein synthesis was not increased in vitro in the soleus muscles taken from these rats. These experiments demonstrate that the anabolic effect of clenbuterol is due in part to beta-adrenergic activity and may involve reduced myofibrillar protein degradation. These results appear to have direct application to nutrition and protein repletion in various catabolic diseases.

Myofibrillar protein breakdown in skeletal muscle progresses through two distinct phases in response to chronic glucocorticoid administration in the rat, i.e., an early phase lasting 4-5 days, during which proteolysis increases followed by a later phase during which proteolysis decreases. The possible involvement of insulin and the iodothyronines in this phenomenon has now been examined. Diabetic, thyroidectomized, and normal rats were treated with corticosteroid for 10-11 days, and at timed intervals muscle proteolysis was evaluated by measuring the release of 3-methyl-L-histidine (3-MH) and tyrosine from the perfused hindquarter as well as the excretion of 3-MH in the urine. Corticosterone (CTC) administration to normal rats increased plasma insulin, whereas plasma 3,5,3'-triiodothyronine responded with an early rise followed by a fall after 4-5 days. However, the biphasic response of myofibrillar proteolysis to chronic glucocorticoid treatment was not abolished in CTC-treated diabetic or thyroidectomized rats. CTC treatment increased release of tyrosine by perfused muscle of diabetic rats but, unlike 3-MH release, did not diminish later. Thus the adaptation of myofibrillar proteolysis to chronic glucocorticoid treatment appears to be independent of insulin and thyroid hormones. However, insulin may play a role in curtailing glucocorticoid-induced breakdown of nonmyofibrillar proteins.

The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.

The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle.

Muscles from fed or 72-hour fasted rats were incubated in the presence of plasma from septic rats, recombinant interleukin 1 alpha (rIL-1 alpha), or recombinant tumor necrosis factor alpha (rTNF alpha), and breakdown of total and myofibrillar protein was assessed by determining release of tyrosine and 3-methylhistidine, respectively. Septic plasma stimulated total protein breakdown in muscles from 72-hour fasted rats by 10% to 20%, while myofibrillar protein breakdown was not affected. When septic plasma was added to muscles from fed rats, neither tyrosine nor 3-methylhistidine release was altered. Various concentrations of recombinant interleukin 1 alpha or recombinant tumor necrosis factor alpha did not affect total or myofibrillar protein breakdown. Since septic plasma did not stimulate myofibrillar protein breakdown, the role of a circulating factor for muscle proteolysis during sepsis remains unclear.

The 20-hr swimming increased the contants of protein, thyrosine and 3-methylhistidine (also per 1 g of protein) in skeletal muscle of rat within 2-24 hrs. A significant increase in 3-methylhistidine excretion followed this on the 2nd day of the recovery period. The revealed combination of changes suggests a simultaneous augmentation of both synthesis and disintegration of protein in muscles after their activity. Hence, the increased excretion of 3-methylhistidine during recovery period reflects an intensive renewal of the molecular content of proteins of actinomyosine complex. The 3-methylhistidine level in intestine increased only for a short time and seems not to contribute to the delayed increase in excretion of the amino acid.

The influence of Ca2+ on myofibrillar proteolysis was evaluated in the isolated extensor digitorum longus muscle incubated in vitro with agents previously shown to increase the intracellular concentration of Ca2+. Myofibrillar proteolysis was evaluated by measuring the release of N tau-methylhistidine, and total proteolysis was evaluated by measuring tyrosine release by incubated muscles after the inhibition of protein synthesis with cycloheximide. Incubated muscles released measurable quantities of N tau-methylhistidine, and muscle contents of the amino acids remained stable over 2 h of incubation. The release of N tau-methylhistidine by incubated muscles was similar to its release by perfused rat muscle in response to brief starvation, indicating the integrity of the incubated muscles. Ca2+ ionophore A23187, dibucaine, procaine, caffeine and elevated K+ concentration increased lactate release by incubated muscles and decreased tissue contents of ATP and phosphocreatine to varying degrees, indicating the metabolic effectiveness of the agents tested. Only A23187 and dibucaine increased total cell Ca2+, and they increased tyrosine release. Caffeine and elevated [K+] increased neither cell Ca2+ nor tyrosine release; however, only A23187 and dibucaine increased tyrosine release significantly. On the other hand, these agents were without effect on myofibrillar proteolysis as assessed by N tau-methylhistidine release by incubated muscles and changes in tissue contents of the amino acid. In fact, some of the agents tested tended to decrease myofibrillar proteolysis slightly. These results indicate that acute elevation of intracellular Ca2+ is associated with increased breakdown of non-myofibrillar but not myofibrillar proteins. Because of this, the role of elevated Ca2+ in muscle atrophy in certain pathological states is questioned. The data also indicate that the breakdown of myofibrillar and non-myofibrillar proteins in muscle is regulated independently and by different pathways, a conclusion reached in previous studies with perfused rat muscle.

The rate of muscle protein synthesis in patients with myotonic dystrophy has been studied, and results correlated with total muscle mass. Whole body and skeletal muscle protein synthesis were estimated by stable isotope methodology with a primed, continuous infusion of 1-[13C]leucine with measurement of incorporation of [13C]leucine into muscle protein in biopsy samples. Whole body leucine flux, protein synthesis, and protein breakdown were only slightly depressed, but muscle protein synthesis was markedly decreased, in myotonic dystrophy. This depression of muscle protein synthesis in myotonic dystrophy correlates with previous observations of impaired insulin-induced muscle uptake of amino acids and supports the suggestion that muscle wasting in this disease is the consequence of defective anabolism in muscle.

The skeletal muscle content of 3-methylhistidine has been measured in two fetuses and four infants of 15 to 38 wk of gestation. The average concentration of skeletal muscle 3-methylhistidine is 1.11, 2.64, and 3.28 (mumol/mixed protein) for fetuses of 15 to 18 of gestation and infants of 26 to 32 and 37 to 38 wk of gestation, respectively. Myofibrillar protein degradation has been measured by the rate of 3-methylhistidine excretion in premature infants suffering from respiratory distress and weighing between 1310 and 2420 g. In 26 balance studies in six infants, total muscle protein breakdown varied from 0.92 to 1.58 g day-1 kg-1 body weight. Calculated fractional catabolism of myofibrillar protein varied from 0.38 to 1.07% per day. A trend toward a higher rate of myofibrillar protein degradation is noted during the infants' acute illness and during their rapid growing phase.

To examine the role of lysosomes in the degradation of skeletal-muscle myofibrillar proteins, we measured the release of N tau-methylhistidine from perfused muscle of starved and fed rats in the presence or absence of agents that inhibit lysosomal proteinase activity. After 1 day of starvation, the release of N tau-methylhistidine by perfused muscle of 4-, 8- and 24-week-old rats increased by 322, 159 and 134% respectively. On the other hand, total protein breakdown, assessed by tyrosine release, increased by 62, 20 and 20% respectively. Inhibitors of lysosomal proteinases as well as high concentrations of insulin or amino acids failed to diminish the release of N tau-methylhistidine by perfused muscle of starved and fed rats, despite a 25-35% inhibition of total protein breakdown. The data strongly suggest that the complete breakdown of myofibrillar proteins occurs via a non-lysosomal pathway. They also suggest that total proteolysis, which primarily reflects non-myofibrillar protein breakdown, occurs at least in part within lysosomes.

1. Myofibrillar protein degradation has been measured by the rate of 3-methylhistidine excretion in premature infants weighing between 635 g and 1295 g. Analyses were made in conjunction with 1--3 day nitrogen balance studies. 2. In 56 balance studies in 36 infants, total muscle protein breakdown varied between 0.70 and 2.58 (mean 1.05) g day-1 kg-1 body weight while the percentage of total muscle protein degraded each day was between 3.3 and 8.3 (mean 4.8). 3. Both total and fractional rates of protein breakdown showed highly significant negative correlations with nitrogen retention but no relationship to total energy input. 4. Protein degradation was higher than average in infants who were losing weight at the time of the balance study, lower in infants who were gaining weight and higher in those who died within 2 weeks of the analysis. 5. Myofibrillar protein breakdown was not different between infants fed orally and those receiving total parenteral nutrition. 6. Generally the effects of nitrogen and evergy status on muscle protein degradation in the premature infants are different from changes reported in adult human beings or adult rats. We suggest that this difference may be a consequence of the very limited energy reserves of the premature infant.

Myofibrillar protein breakdown during brief and prolonged starvation was assessed in perfused rat skeletal muscle from 8-week-old fat-fed rats that conserve skeletal muscle protein during starvation and survive for 12 to 15 days and age-matched chow-fed rats that do not conserve protein and survive only five to six days. Following the inhibition of protein synthesis with cycloheximide, myofibrillar proteolysis was assessed by measuring the release of 3-methylhistidine from the perfused rat hindquarter while simultaneous measurement of total protein breakdown was assessed by measuring tyrosine release. Myofibrillar proteolysis progressed through three distinct phases during starvation: an early phase occurring within 24 hours in which proteolysis increased in all rats, a middle phase, which took three to five days to develop and during which proteolysis decreased and was present only in fat-fed rats, and a late phase in which proteolysis again increased. Total protein breakdown (ie, tyrosine release) changed little in phase I, decreased in phase II, and increased in phase III. The release of 3-methylhistidine from the perfused hindquarter reflected changes in muscle and urine of intact rats suggesting that data obtained with the perfused hindquarter reflected the in vivo situation. Insulin, amino acids, high concentrations of glucose, indomethacin, or epinephrine as well as adrenalectomy failed to attenuate the increase in 3-methylhistidine release from the perfused hindquarter during brief and late starvation. Free fatty acids and ketone bodies were also without effect in vitro. Refeeding fasting rats for four hours decreased myofibrillar proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The recoveries of radioactivity in cattle urine following the intravenous administration of N tau-[14CH3]methyl histidine were essentially quantitative in 5--7 d in non-lactating cows, bulls and steers and did not change with age. 2. The N tau-methyl histidine was excreted unchanged in urine. 3. N tau-methyl histidine occurred in muscle extracts both in the free form and as a perchloric acid-soluble, acid-labile form which accounted for approximately 85% of the total non-bound N tau-methyl histidine in muscle and appeared identical to a similar component identified in muscle extracts of sheep and pigs. 4. There was probably an age-related decrease in the concentration of the acid-labile component in muscle but which did not produce a measurable change in recovery of radioactivity in urine. 5. The daily excretion of N tau-methyl histidine (E, mumol) by male cattle was highly correlated with live weight (W, kg) by the equation: E = 50 . 4 + 3 . 536 (+/- 0 . 044)W (r 0. 997). The excretions progressively decreased from 4 . 04 mumol/d per kg at 100 kg weight to 3 . 62 mumol/d per kg at 600 kg. 6. By the criterion of the rate of clearance of labelled N tau-methyl histidine from the body, the excretion of N tau-methyl histidine in urine appears to be a valid index of muscle protein breakdown in cattle.

Excreta were collected for four consecutive days from 4- to 18-week-old cockerels following subcutaneous injection of N tau-[14CH3]methylhistidine. The recoveries of radioactivity in excreta were incomplete and progressively decreased with increasing age. Most of the radioactivity not recovered in excreta after 4 d was found in skeletal muscle where greater than 55% of the radioactivity present was in the N tau-methylhistidine-containing dipeptide, balenine. This peptide appeared to be relatively stable so that most of the labelled N tau-methylhistidine incorporated was not released during the period of the recovery measurements. The total pool of non-protein bound N tau-methylhistidine (free (free N tau-methylhistidine + balenine) in pectoral and mixed thigh muscles increased with age and relative to the daily excretion of N tau-methylhistidine. At 18 weeks the pool was 3.3 times the daily excretion of N tau-methylhistidine. These observations account for the decreasing recoveries of radioactivity in excreta described previously, due to progressive dilution of labelled N tau-methylhistidine in an expanding pool of non-protein-bound N tau-methylhistidine, part of which was relatively stable. It is concluded that excretion of N tau-methylhistidine by 4- to 18-week-old cockerels cannot be used as a reliable index of muscle protein breakdown in vivo.

We used the perfused hemicorpus preparation to measure individual rates of protein synthesis and degradation. Using fed animals, perfused either with or without insulin, muscle protein synthesis and hemicorpus protein degradation rates were similar, but myofibrillar protein degradation was clearly increased in the uremic preparations. When the animals were fasted, differences in the rates of skeletal muscle protein turnover were apparent. Uremic rats lost more wt at both 24 and 48 hr of fasting when compared to either ad libitum fed or pair-fed controls who started fasting at body wts equivalent to our uremic rats. The accelerated wt loss was accompanied by lower rates of protein synthesis, higher degradation rates, and greater net protein catabolism in our uremic rats. Alterations in body lipid content were present in uremia and correlated with the rate of protein degradation in both control and uremic rats. These data demonstrated that even in the fed state, uremia is associated with subtle alterations in skeletal muscle protein turnover. When stressed, these alterations become more pronounced. Insufficient stores of body lipids, either due to inadequate nutrition or altered metabolism, may contribute to the alterations in muscle protein turnover seen in chronic renal insufficiency.

The rate of protein degradation is usually thought to be first order, i.e. determined by the nature of the protein as a substrate. It is not immediately apparent if this is the case for the overall process in the cell since rates of turnover of individual proteins may vary between tissues. In muscle the characteristics of protein turnover in relation to DNA-unit size have led to the development of a model for protein turnover in which degradation rates are determined by the rate of dissociation of protein subunits from the myofibrillar matrix. This is a necessary step if heterogeneous turnover occurs and if degradation and resynthesis of myofibrillar proteins occurs peripherally to the myofibril. As a result a first order rate can be envisaged so that during muscle growth the protein mass per unit DNA increases to a characteristic amount thus determining the specific activity of the degrading system. Such a mechanism may apply to all cells.

Myofibrillar protein breakdown was evaluated by measuring the release of N tau-methylhistidine by isolated rat skeletal muscles or perfused rat muscles in the presence of a variety of agents known to affect Na+ flux. Total cell proteolysis was evaluated simultaneously by measuring tyrosine release by muscles after the inhibition of protein synthesis with cycloheximide. Treatment of muscles with the Na+ ionophore monensin or inhibitors of Na+-K+ ATPase (ouabain, digoxin or vanadate) decreased N tau-methylhistidine release by muscles by 21-35%. A phorbol ester (phorbol 12-myristate 13-acetate) as well as a synthetic diacylglycerol known to activate protein kinase C and a Na+/H+ antiport also decreased N tau-methylhistidine release by muscles. Removal of extracellular Na+ blocked the ability of these agents to attenuate N tau-methylhistidine release by muscles, suggesting that their effectiveness required a change in Na+ flux. In contrast with N tau-methylhistidine release by muscles, these agents, except for monensin, did not effect the release of tyrosine, suggesting that they attenuate specifically the breakdown of myofibrillar proteins. Overall these results indicate a link between Na+ and the regulation of protein breakdown in rat skeletal muscle, whereby an influx of Na+ can result in a decrease in myofibrillar proteolysis. Left unresolved is whether phospholipid hydrolysis is involved in this scheme.